Phylogenetically Conserved Peritoneal Fibrosis Response to an Immunologic Adjuvant in Ray-Finned Fishes

Home , Bleeding heart tetra, Centrarchiformes, Dichotomyctere, Peacock gudgeon, Schistocephalus solidus, Sphaeramia

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1 PHYLOGENETICALLY CONSERVED PERITONEAL FIBROSIS RESPONSE TO 2 AN IMMUNOLOGIC ADJUVANT IN RAY-FINNED FISHES 3 4 Running title: Evolution of peritoneal fibrosis in fish 5 6 Authors: Milan VRTÍLEK1*, Daniel I. BOLNICK2 7 8 Affiliations: 9 1The Czech Academy of Sciences, Institute of Vertebrate Biology, Květná 8, 603 65 Brno, Czech 10 Republic 11 2Department of Ecology and Evolutionary Biology, 75 N. Eagleville Road, Unit 3043, University 12 of Connecticut, Storrs, Connecticut 06269, USA 13 14 *Corresponding author: The Czech Academy of Sciences, Institute of Vertebrate Biology, 15 Květná 8, 603 65 Brno, Czech Republic; email: [email protected] 16 17 Author contributions: DIB and MV designed the study, MV conducted experimental work, 18 performed data collection and analysis, MV and DIB wrote the manuscript. 19 20 Acknowledgements: We would like to thank to Katherine R. Lewkowicz, Meghan F. 21 Maciejewski, Lauren E. Fuess, Amanda K. Hund, Mariah L. Kenney, Foen Peng and Stephen P. 22 De Lisle (members of the Bolnick Lab, University of Connecticut) for their help throughout the 23 fish experiment and discussions on peritoneal fibrosis. Comments by Amanda K. Hund, Martin 24 Reichard, Jakub Žák, Radim Blažek, Markéta Ondračková and Matej Polačik (Institute of 25 Vertebrate Biology, CAS) helped to improve the manuscript. We are thankful to Quinebaug 26 Valley State Fish Hatchery (CT, USA) for providing rainbow trout. MV’s stay at the University 27 of Connecticut was supported by Fulbright Commission fellowship for research scholars. The 28 project was funded by NIH project (NIAID grant 1R01AI123659-01A1) held by DIB. The 29 experimental work was approved by University of Connecticut, Protocol No. A18-0008. 30 31 Data Accessibility Statement: Data will be made publicly available after acceptance along with 32 the statistical code under DOI: 10.6084/m9.figshare.12619367. 33 34 Conflict of interest: The authors have declared no conflict of interest. 35 36 Word count: 4498 (2 Tables and 2 Figures) 1

37 ABSTRACT

38 Antagonistic interactions between hosts and parasites may drive the evolution of novel host

39 defenses, or new parasite strategies. Host immunity is therefore one of the fastest evolving traits.

40 But where do the novel immune traits come from? Here, we test for phylogenetic conservation in

41 a rapidly evolving immune trait – peritoneal fibrosis. Peritoneal fibrosis is a costly defense

42 against novel specialist tapeworm Schistocephalus solidus (Cestoda) expressed in some

43 freshwater populations of threespine stickleback fish (Gasterosteus aculeatus, Perciformes). We

44 asked whether stickleback fibrosis is a derived species-specific trait or an ancestral immune

45 response that was widely distributed across ray-finned fish (Actinopterygii) only to be employed

46 by threespine stickleback against the specialist parasite. We combined literature review on

47 peritoneal fibrosis with a comparative experiment using either parasite-specific, or non-specific,

48 immune challenge in deliberately selected species across fish tree of life. We show that ray-

49 finned fish are broadly, but not universally, able to induce peritoneal fibrosis when challenged

50 with a generic stimulus (Alum adjuvant). The experimental species were, however, largely

51 indifferent to the tapeworm antigen homogenate. Peritoneal fibrosis, thus, appears to be a

52 common and deeply conserved fish immune response that was co-opted by stickleback to adapt

53 to a new selective challenge.

55 KEYWORDS: Actinopterygii, comparative experiment, immunity, peritoneal fibrosis,

56 stickleback, vaccination

58 INTRODUCTION

59 The comparative immunology research makes it clear that many of the fastest-evolving and most

60 polymorphic genes in vertebrates are involved in immunity (Litman and Cooper 2007; Lazzaro

61 and Clark 2012; Slodkowicz and Golman 2020). Most notable is the evolutionary reshuffling of

62 the genes coding Toll-like receptors (TLR) (Solbakken et al. 2017; Velová et al. 2018), or the

63 diversity of major histocompatibility complex (MHC) genes (Malmstrøm et al. 2016; Radwan et

64 al. 2020). Conversely, the broad outlines of innate immunity are ancient, such as one of the most

65 ancestral immune cytokines, transforming growth factor β (TGF-β), which seems to be conserved

66 across the animal kingdom (Herpin et al. 2004). And yet, even some highly conserved immune

67 genes and processes have been lost or changed past recognition in certain vertebrate clades, such

68 as the loss of MHCII genes in the Atlantic cod (Malmstrøm et al. 2013). This contrast between

69 deep evolutionary conservation, and rapid co-evolutionary dynamics, is puzzling. What features

70 of the immune system are highly conserved, and what are evolutionarily labile?

71 Vertebrates possess, in principle, two functionally distinct strategies combining innate and

72 adaptive immunity to cope with infection according to parasite type (Flajnik and Du Pasquier

73 2004; Allen and Maizels 2011). Type 1 immune response is triggered by fast reproducing

74 pathogens, as microbes, with the aim to quickly eliminate the infection through pro-inflammatory

75 trajectory (Allen and Maizels 2011). On the other hand, type 2 immune response is typically

76 directed to reduce the effect of a multicellular parasite, such as a helminth worm, by containment

77 and encapsulation (Allen and Maizels 2011; Gause et al. 2013). Type 2 immunity largely shares

78 signaling pathways with tissue repair and wound healing (Gause et al. 2013; Thannickal et al.

79 2014). Perpetual tissue damage and wound healing may, however, result in excessive

80 accumulation of fibrous connective matter called fibrosis (Thannickal et al. 2014). This fibrosis

81 has been found to effectively suppress growth of certain parasites, or even lead to parasite death

82 (Weber et al., in preparation). However, the benefits of tissue repair and parasite containment can

83 come with a cost from chronic type 2 immune response during persistent or recurrent infections,

84 which may develop into serious health issues or even death (Gause et al. 2013; De Lisle and

85 Bolnick 2020). Here, we measure the extent of evolutionary conservation of a key immune

86 phenotype, fibrosis.

87 Recent findings on inter-population variation in helminth resistance from threespine

88 stickleback fish (Gasterosteus aculeatus) demonstrate that anti-helminthic fibrosis response is a

89 fast-evolving immune trait (Weber et al. 2017a). Stickleback, originally a marine species, has

90 only recently invaded freshwater habitats where it experienced greater risk of acquiring parasitic

91 tapeworm Schistocephalus solidus (Cestoda) through feeding on freshwater copepods (Barber

92 and Scharsack 2010; Rahn et al. 2016). When ingested, the tapeworm larva migrates through the

93 intestinal wall to the peritoneal cavity of the fish and grows to its final size, often >30% the

94 host’s mass (Arme and Owen 1967; Ritter et al. 2017). The threespine stickleback is the obligate

95 intermediate host of this specialized parasite. Some populations of stickleback have evolved a

96 capacity to suppress S. solidus growth by encapsulating it in fibrotic tissue, sometimes leading to

97 successfully killing and eliminating the parasite (Weber et al. 2017b).

98 This presumably beneficial form of resistance has costs in greatly suppressing female

99 gonad development and male reproduction (De Lisle and Bolnick 2020; Weber et al., in

100 preparation). These costs may explain why the intensive peritoneal response to S. solidus has

101 evolved in only some lake populations, and in some geographic regions of the sticklebacks’ range

102 (Weber et al. 2017a). In the other populations, stickleback have apparently adopted a non-fibrotic

103 tolerance response to reproduce despite infection (Weber et al. 2017a). These non-fibrotic

104 populations exhibit active up-regulation of fibrosis-suppression genes in response to cestode

105 infection (Lohman et al. 2017; Fuess et al. 2020). The ancestral marine populations come very

106 rarely into contact with S. solidus which does not hatch in saline water (Barber and Scharsack

107 2010), and do not exhibit observable fibrosis in the wild or in captivity (Hund et al. 2020). These

108 various marine and freshwater populations have been diverging only since Pleistocene

109 deglaciation (~12,000 years), indicating that their fibrosis response has evolved surprisingly

110 quickly for such a fundamental immune process.

111 The peritoneal fibrosis can reliably be provoked in both fibrotic and non-fibrotic

112 populations of stickleback by a generalized immune challenge (injection with a non-specific

113 Alum adjuvant) (Hund et al. 2020), while only the resistant populations initiate fibrosis in

114 response to tapeworm protein injection. So, the physiological capacity to initiate fibrosis seems

115 conserved in stickleback and its sensitivity to the tapeworm fast-evolving. We therefore wished

116 to determine whether this peritoneal fibrosis is similarly labile, or conserved across a broader

117 range of fish species. One possibility is that peritoneal fibrosis is unique to stickleback, which is

118 the only fish species to host S. solidus, an unusual parasite in that it invades the fish peritoneal

119 cavity (Barber and Scharsack 2010). Or, fibrosis may be a widely conserved trait that stickleback

120 have uniquely co-opted to deal with this specialist parasite species. Is then peritoneal fibrosis an

121 ancestral character state dating back to the origin of teleosts, or beyond?

122 To address this question, we begin with a broad literature review of fibrosis in fishes to

123 systematically summarize documented instances for the first time. Then, we present an

124 experimental test of how widely ray-finned fish (Actinopterygii) exhibit peritoneal fibrosis

125 response towards an immune challenge based on a recent experimental study of fibrosis response

126 in stickleback (Hund et al. 2020).

127

128

129 METHODS

130 Literature review

131 We performed search for articles mentioning fibrosis or encapsulation from Actinopterygii using

132 Web of Science database. We then sorted the collected suitable articles according to the presence

133 of fibrosis or encapsulation, its topology and cause. We also extracted the taxonomic information

134 and present the results in a pivot table. For detailed methods, please see Appendix.

135

136 Species selected for the experiment

137 To conduct a phylogenetically broad assay of peritoneal fibrosis response in ray-finned fishes

138 (Actinopterygii), we deliberately chose a diverse set of species spread across the phylogenetic

139 tree of Actinopterygii. We experimentally vaccinated 17 species of fish (Table 1). These species

140 were chosen to achieve broad phylogenetic diversity, but were restricted to commercially

141 available small fish (body length of 1-4”, or 2-10 cm, and live weight 0.2-6.0 g). We obtained

142 most of the fish from a fish reseller. Local trout hatchery donated fingerlings of rainbow trout

143 (Oncorhynchus mykiss). We received eggs of the turquoise killifish (Nothobranchius furzeri,

144 population MZCS 222) from a stock retained at the Institute of Vertebrate Biology CAS in Brno,

145 Czech Republic. We hatched and maintained the killifish according to breeding protocol (Polačik

146 et al. 2016) until they reached two months when fully mature. We also included wild-caught

147 threespine stickleback (Gasterosteus aculeatus) that came from two distinct populations (Loch

148 Hosta and Loch a’ Bharpa), both originating from North Uist, Scotland, UK, provided by Andrew

149 MacColl (University of Nottingham, UK) and maintained at our housing facility for six months

150 before the experiment.

Table 1. Species selected for the experimental test.

151

152 Fish housing

153 We standardized housing conditions across most of the species. We planned to have 5-7

154 individuals per species per treatment. We prepared 20-gal (~76 L) tanks with reverse-osmosis

155 (RO) water conditioned to conductivity between 700-800 µS/cm with sea salt (Instant Ocean®).

156 We adjusted salinity for marine species to 35 mg/kg. Each tank contained air driven sponge filter

157 and heater with temperature set to 25 °C (water temperature ranged between 24.5-26 °C). We

158 also provided a seaweed-like plastic shelter for fish. We fed fish every morning with frozen

159 bloodworms (Chironomidae), mysis shrimp (Mysidae), or dried sushi nori seaweed (Pyropia sp.)

160 according to species-specific diet requirements. We held the rainbow trout at water temperature

161 of 12 °C as our standard temperature would be stressful to them. Similarly, stickleback are

162 sensitive to high temperatures and we therefore kept them in their original recirculation system at

163 19 °C and 1900 µS/cm throughout the treatment. All the other fish were habituated to the

164 common tank setup for at least five days before treatment.

165

166 Reagents

167 Drawing on a recent experimental study of fibrosis response in stickleback (Hund et al. 2020), we

168 challenged selected fish species with a generalized immune stimulant (Alum vaccination

169 adjuvant), or with antigens from a specialist helminth parasite (S. solidus) that induces peritoneal

170 fibrosis in some populations of threespine stickleback. Saline solution served as a control. All

171 three treatments were delivered via peritoneal injection (the site of S. solidus infection), for all 17

172 species.

173 The control treatment was an injection of 1X phosphate-buffered saline (PBS) (20 µL per 1

174 g of species average of live weight), which was also the solution for delivering the other

175 treatments. The second treatment consisted of tapeworm antigen homogenate (abbreviated TH)

176 suspended in PBS. Hund et al. (2020) showed that injection of 9 mg of TH per 1kg live fish mass

177 (0.009 mg/g) induced rapid fibrosis in tapeworm-resistant stickleback populations. We obtained

178 S. solidus tapeworms dissected from wild-caught threespine stickleback (Gosling Lake,

179 Vancouver Island, BC, Canada). We used two tapeworms to prepare the homogenate, sonified

180 them in PBS on ice and then centrifuged the suspension at 4000 RPM at 4 °C for 20 minutes. We

181 assessed overall protein concentration in the upper fraction of the solution using RED 660TM

182 protein assay (G-Biosciences) measured in triplicates and then diluted the sample to 0.45 mg/mL.

183 We aimed at injecting 20 µL of the solution per 1 g of live fish weight and to obtain the desired

184 dose 0.009 mg of tapeworm protein for 1 g of fish weight (or 9 mg/kg, as in Hund et al. (2020)).

185 We then aliquoted the homogenate in 0.6-ml Eppendorf tubes and stored at -20 °C for later

186 injections. Using the tissue of S. solidus, we wanted to test whether the stickleback response to

187 this specialized parasite is unique among other fishes. In principle, for instance, it is possible that

188 S. solidus protein contains distinctive antigens that any fish species would recognize as foreign.

189 The third treatment was a 1% Alum solution (20 µL per 1 g of species average of fish live

190 weight). Alum promotes activation of innate immune response and is commonly used as a

191 vaccine adjuvant (Kool et al. 2012). We dissolved 2% AlumVax Phosphate (OZ Biosciences) in

192 1:1 with PBS. This concentration of Alum induces peritoneal fibrosis in marine and freshwater

193 stickleback population irrespective of their tapeworm resistance (Hund et al. 2020).

194

195 Injections

196 At their arrival to our fish facility, we weighed each fish species on a balance to 0.01 g (Gene

197 Mate GP-600) to estimate total volume of solution to be injected per individual (based on species

198 average live mass). We injected 20 µL of the solution per 1 g of average live weight with Ultra-

199 Fine insulin syringes. We always filled syringes aseptically under laminar flow cabinet 1-2 days

200 before the injections, stored them at 4 °C and used 1 syringe per individual. Prior work (Hund et

201 al. 2020) confirmed that syringes prepared in this way were effective at inducing fibrosis. We

202 injected fish peritoneally through their left flank after anesthesia with MS-222 (200 mg/L for up

203 to two minutes). Fish were then allowed to recover from anesthesia in a highly aerated tank

204 water. We returned them to their original tank once they were swimming upright, but typically

205 after more than five minutes after the injection. The different treatment groups were held in

206 separate tanks because peritoneal fibrosis is a specific response to the treatment unaffected by

207 common housing conditions (Hund et al. 2020).

208

209 Dissections and scoring fibrosis level

210 We euthanized fish five days post-injection, using an overdose of MS-222 (500 mg/L, > 5 min).

211 Because trout and stickleback were kept at lower temperatures, which may slow immune

212 responses (Rijkers et al. 1980), we dissected trout 10 days after injection, and stickleback at both

213 5 and 10 days. We also dissected 1-2 individuals from each species prior the injections to

214 examine species-specific anatomy and assess the baseline level of peritoneal fibrosis before

215 injections. We dissected fish immediately after euthanasia under stereo-microscope,

216 photographed and scored their level of fibrosis, using an ordinal categorical score. The peritoneal

217 fibrosis score ranged between 0 and 3. Zero represents the absence of noticeable fibrosis, where

218 the internal organs (liver, intestine, gonads) move freely apart from each other and from the

219 peritoneal wall when moved with tweezers. Level 1 was for organs adhered together forming an

220 interconnected conglomerate that moves as a unit. Level 2 was scored when the internal organs

221 also attached to the peritoneal wall, but it was still possible to free them. Level 3 was the extreme

222 form of organ adhesion where the peritoneal lining tore apart and remained attached to the organs

223 after the body cavity had to be forcibly opened. Note that peritoneal fibrosis levels 1, 2, and 3

224 used here correspond with levels 2, 3, and 4, respectively, used by Hund et al. (2020). For

225 illustration of the 0-4 scale (Hund et al. 2020) see video at https://youtu.be/yKvcRVCSpWI. We

226 also recorded total and dissected weight of the dead fish (0.001 g, Sartorius Element ELT202),

227 fish sex (where it could be determined), and the presence of any internal parasites.

228 We excluded two species before data analysis. The clown feather-back (Chitala ornata)

229 were already extremely fibrosed (level 3) when they arrived to our facility due to unknown

230 reason and the kissing gourami (Helostoma temminckii) developed white spot disease during the

231 experiment after they were injected. This made the total number of species tested 17 (Table 1).

232

233 Data analysis

234 We formally tested the interaction between the effect of treatment (PBS, TH, Alum) and

235 experimental species identity on individual fibrosis score using generalized least squares (GLS)

236 model (function gls, library “nlme” v.3.1-148, Pinheiro et al. 2018). The response variable was

237 fibrosis level (ordered integers 0-3) scored from the individual’s left flank. We set treatment,

238 species and their interaction as fixed model effects. We attempted to originally analyze the

239 ordinal response variable using Cumulative link models (CLM, function clm, library “ordinal”

240 v.2019.12-10, Christensen 2019), but CLM with treatment–species interaction failed to converge

241 due to model singularity (e.g., too many groups with zero variance because all individuals had

242 identical fibrosis scores).

243 To assess phylogenetic signal in the experimental data, we created a species tree based on

244 the recent comprehensive phylogeny of ray-finned fishes by Hughes et al. (2018) with estimated

245 divergence times. We then obtained phylogenetic signal (i.e. the tendency of related species to

246 show similar response) and ancestral character state for two species “traits”: the maximum level

247 of peritoneal fibrosis in a species and species relative response in the positive control (Alum)

248 compared to negative control (PBS). The species relative response accounts for some species

249 having basic fibrosis level 1 while 0 was observed in the majority (12/17 species; Fig. 1). We

250 measured phylogenetic signal with Pagel’s λ (function phylosig, with method specification to

251 “lambda”, library “phytools” v.0.7-47 (Revell 2012)). We then estimated ancestral character state 11

252 with maximum likelihood approach using function anc.ML (specifying Brownian motion mode

253 of evolution, library “phytools”). All analyses were performed in R software v. 4.0.1 (R Core

254 Developmental Team 2019).

255

256

257 RESULTS

258 Literature review: Peritoneal fibrosis in fish is known but not well documented

259 Our first aim was to gather an overview of publication record encompassing peritoneal fibrosis in

260 fish. In the articles we collected (for detailed methods see Appendix), general fibrotic response

261 was documented from a wide array of ray-finned fish (Actinopterygii). We found 375 out of the

262 1335 articles (i.e. 28%) to be suitable for our study (i.e. articles mentioning fibrosis or

263 encapsulation from Actinopterygii). The most-represented species came from the orders

264 Cypriniformes (61 [including articles with multiple species]; 16% of relevant articles) and

265 Salmoniformes (51; 14%) (Table 2). Authors typically identified signs of fibrosis during an

266 autopsy, though some cases were observed from fish integument as well (e.g. capsules of skin

267 parasites). Most of the suitable articles reported parasitism (197; 53%) or treatment (81; 22%) as

268 the cause. Overall, fibrosis was present either on its own (175; 47%) or in combination with

269 encapsulation of a foreign object (22; 6%).

Table 2. Overview of the literature search.

270

271 The extent of fibrosis was usually described only qualitatively, along with the identity of

272 the affected organs or tissues. Taking articles related to fibrosis (reporting fibrosis alone or in

273 combination with encapsulation, 198 articles), its incidence was mainly confined to visceral

274 organs (152 cases, 77%). Fibrosis located around or in the internal organs was, however, mainly

275 interstitial fibrosis often represented by tissue scarification after damage. We found only 7

276 articles that were dealing with peritoneal fibrosis specifically. These seven articles are diverse

277 with regard to species taxonomic position and the cause of the fibrosis response (e.g., tapeworm

278 infection, vaccination, radio-transmitter implantation) (Table 2). From this literature survey, we

279 conclude that fibrosis is known in a number of fish species. Yet, the peritoneal fibrosis is very

280 scarcely reported which limits our ability to draw broader conclusions about its evolutionary

281 history or its function.

282

283 Comparative immunological experiment: Common and strong immune response to Alum

284 We conducted a phylogenetically structured experimental study of ray-finned fish peritoneal

285 fibrosis in response to immune challenge to reach more systematic understanding of its evolution.

286 The level of fibrosis differed between experimental treatments and across species (GLS,

287 treatment-species interaction, F34,251 = 7.93, P < 0.001) (Fig. 1). For most species, we observed

288 no detectable or low fibrosis in control-injected (PBS) fish as well as in the tapeworm

289 homogenate (TH) treatment (Fig. 1). However, there were two species, the common carp

290 (Cyprinus carpio) and the channel catfish (Ictalurus punctatus), with variable individual response

291 both in the PBS control and TH treatment (Fig. 1). Also note that in some species (like D. rerio,

292 S. fasciatus, C. viridis), the default fibrosis level was 1 (organs attached together).

Figure 1. Peritoneal fibrosis in the experimental fish species.

293

294 In contrast to the negative control and TH, the positive control (Alum injection) induced

295 strong peritoneal fibrosis in most of the species (15 of 17). The response was typically high to

296 extreme (fibrosis level 2 or 3, Fig. 1). The exceptions were two species of tetras, the Mexican

297 tetra (Astyanax mexicanus) and the bleeding-heart tetra (Hyphessobrycon erythrostigma), which

298 did not respond to any of the treatments (Fig. 1).

299 Phylogenetic signal for species maximum fibrosis and species relative response to the

300 positive control (average Alum vs. PBS difference) both appeared to be strong, significantly

301 different from random evolution (Pagel’s λ > 0.987 and P < 0.005, for both traits). Ancestral state

302 at the base of the ray-finned fishes, i.e. at the divergence between the Senegal bichir (Polypterus

303 senegalus) and the other species from our experiment, was estimated 1.862 for the species

304 maximum fibrosis (Fig. 2) and 1.325 for species’ relative fibrosis response to the Alum treatment

305 (average difference between Alum vs. PBS).

Figure 2. Ancestral state reconstruction of species maximum peritoneal fibrosis.

306

307

308 DISCUSSION

309 This study represents one of the first comparative experimental assays of the macroevolution of

310 an immune response. We focused on evaluating the prevalence of peritoneal fibrosis response

311 across fishes, because this response has been shown to contribute to parasite growth suppression 14

312 and elimination in some recently-evolved post-glacial lake populations of threespine stickleback.

313 We show that published literature contains little data on peritoneal fibrosis in ray-finned fishes.

314 To fill this gap, we experimentally tested the prevalence of peritoneal fibrosis in a wide array of

315 species across the phylogeny of Actinopterygii. Our immune challenge resulted in a variable

316 level of fibrosis between treatments and among species. Response to homogenate from the

317 stickleback-specialized tapeworm was weak at best. This finding confirms that the use of fibrosis

318 pathways in response to S. solidus is a recently-evolved trait. The positive control treatment

319 (Alum), on the other hand, provoked strong peritoneal fibrosis in most of the species tested

320 except one specific lineage – two species of tetras (from family Characidae, Characiformes). The

321 results therefore suggest that, despite being rarely observed or reported, the capacity for

322 peritoneal fibrosis is a phylogenetically widespread aspect of fish immunity. Ancestral character

323 state reconstruction indicates that the peritoneal fibrotic response is phylogenetically conserved at

324 least to the origin of ray-finned fishes (Fig. 2), estimated around 380 million years ago (Hughes

325 et al. 2018).

326

327 Lack of peritoneal fibrosis in publications across ray-finned fish

328 The literature search indicated that fish initiate fibrosis most frequently in response to tissue

329 injury and/or parasitism. Thus, we can identify two main roles of fibrosis – maintenance of

330 homeostasis in damaged tissue, and formation of physical barrier around an invader

331 (encapsulation). Indeed, Gause et al. (2013) proposed an evolutionary hypothesis for the origin of

332 parasite encapsulation from the ancestral repair response to tissue mechanical damage. The article

333 collection also contained several cases (almost 1/3 of the articles mentioning fibrosis or

334 encapsulation), where parasite encapsulation happened without more wide-spread fibrosis.

335 Our study was motivated by inter-population variation in threespine sticklebacks’ ability to

336 encapsulate parasitic tapeworm S. solidus (Weber et al. 2017b). The inter-population variation

337 probably stems from an evolutionary trade-off between the benefit of resistance (early

338 encapsulation of the worm) and the risk of organ adhesion, excessive fibroblast proliferation, and

339 ultimately partial sterility in the stickleback (Weber et al., in preparation; De Lisle and Bolnick

340 2020). Previous records on peritoneal fibrosis in threespine stickleback are mostly lacking (but

341 see Hoffman 1975, p.175), although the phenomenon is found in numerous populations across

342 the species’ circumpolar range. This is in line with the literature search, where we found only

343 very few studies reporting peritoneal fibrosis in fish. In these few articles, peritoneal fibrosis was

344 associated with serious intrusion of body integrity, e.g., radio-transmitter implantation (Mangan

345 1998), vaccination with bacteria (Colquhoun et al. 1998), or, similarly to the stickleback,

346 tapeworm infection (Abdelsalam et al. 2016). Apparently, the stress has to be intensive and/or

347 chronic to trigger peritoneal fibrosis. The question thus remained whether peritoneal fibrosis is

348 that rare and highly specific response across fish species. We used a phylogenetically informed

349 immune challenge experiment with selected representatives across the fish tree of life to answer

350 this question.

351

352 Peritoneal fibrosis in response to vaccination adjuvant was prevalent in most fish species

353 We successfully triggered peritoneal fibrosis in most of the fish species tested with the positive

354 control (Alum). Alum is a commonly used vaccination adjuvant that causes influx of multiple 16

355 types of immune cells into the injected region and alerts individual’s immune system (Kool et al.

356 2012). Yet, the particular mechanism of how Alum promotes vaccination is still unknown. In the

357 case of peritoneal fibrosis, the Alum crystals may in fact act as an irritating agent that stimulates

358 the type 2 immune response leading to containment of the body non-self (Gause et al. 2013). The

359 widespread response to the Alum injection demonstrates that the capacity for peritoneal fibrosis

360 is distributed across ray-finned fish phylogeny. Absence of fish peritoneal fibrosis in the

361 published literature may thus stem from high specificity (peritoneal cavity invasion), low

362 severity/chronicity of the common stressors, or the phenomenon may simply have been

363 overlooked as was until recently in stickleback.

364

365 The experimental exceptions

366 Individual variation. The homogenate prepared from S. solidus tapeworm caused peritoneal

367 fibrosis only in two tested species and one population of the threespine stickleback. In common

368 carp (Cyprinus carpio) and channel catfish (Ictalurus punctatus), the level of peritoneal fibrosis

369 varied among individuals both in the TH and negative control (PBS) treatments. We recorded

370 similar pattern also in threespine stickleback from Loch Hosta population after 10 days post-

371 injection. The response was comparable between TH and the negative control (PBS). Based on

372 the individual variation and the pre-treatment dissections, it seems like different individuals of

373 the carp, catfish and Loch Hosta stickleback might be more or less sensitive to the injection itself.

374 Individual variation in these three groups contrasts with the largely uniform response exhibited in

375 each treatment by the remaining species.

376 General indifference to the TH treatment. Schistocephalus solidus is a parasite specialist of

377 the threespine stickleback. The cue to trigger peritoneal fibrosis in threespine stickleback in

378 response to the tapeworm is probably specific, as shown by some populations that fail to respond

379 to certain genotypes of S. solidus (Weber et al. 2017a). Parasite community in North Uist

380 stickleback is relatively rich and includes S. solidus (Rahn et al. 2016). The Scottish stickleback

381 were also observed to show peritoneal fibrosis in the wild, with a’ Bharpa population having

382 strong response and Hosta population absent (A. MacColl, pers. comm.). The tapeworm protein

383 effective dose used here (9 mg/g of fish live weight) triggered strong fibrosis in a naturally

384 fibrotic lake population from Vancouver Island (Hund et al. 2020), but it is possible that the

385 Scottish stickleback do not recognize tapeworms from western Canada. The specific cue that

386 triggers peritoneal fibrosis in response to the tapeworm infection is unknown, though presumably

387 protein-based, and the work on its identification currently ongoing. It may still be as well possible

388 that the expression of peritoneal fibrosis might be suppressed by stickleback in some non-fibrotic

389 populations (Lohman et al. 2017; Fuess et al. 2020).

390 Absence of peritoneal fibrosis response. Our experimental data show that peritoneal fibrosis

391 is widespread across fish phylogeny, except for two related species – the Mexican tetra (Astyanax

392 mexicanus) and the bleeding-heart tetra (Hyphessobrycon erythrostigma), which did not respond

393 to any of the treatments. The adaptation of some populations of Mexican tetra to the freshwater

394 cave systems and lower parasitic burden could partially explain the observed pattern (Peuß et al.

395 2020). Taking into account the risks associated with the peritoneal fibrosis described in the

396 threespine stickleback, maintenance of such response could be too costly for the Mexican tetra.

397 Interestingly, the cave Mexican tetras exhibit marked reduction of visceral adipose tissue

398 immunopathology compared to the surface populations (Peuß et al. 2020). However, we provide

399 an evidence that the lack of the peritoneal fibrosis response might be more prevalent among

400 Characiformes: both of the tetra species in our study didn’t respond to Alum. The non-specific

401 immunity of these tetra species might therefore differ from the other ray-finned fish, and is a

402 tempting target for more research, though we cannot yet generalize to many populations of each

403 species, or across the entire clade. The family Characidae containing the two tetras consists of

404 over a thousand species widely distributed in fresh waters from Texas, USA to Argentina. It

405 would be interesting to uncover the mechanism for and phylogenetic extent of the absence of

406 peritoneal fibrosis in more detail. Interestingly, our literature search indicates that Characiformes

407 are able to encapsulate parasites and also show signs of (interstitial) tissue fibrosis (Table 2).

408

409 Conclusion

410 As proposed by Gause et al. (2013), fibrosis is probably an ancient trait evolved from wound

411 healing; the formerly repairing mechanism now suits also coping with endoparasites. We showed

412 that, despite being rarely reported in the published literature, the potential to develop peritoneal

413 fibrosis is widespread across fish phylogeny and it can be triggered through a general treatment

414 (Alum peritoneal injection) in almost all tested fish. The comparative immunology experiment,

415 such as the one we performed, is particularly powerful and broad approach to infer historical

416 origin, evolutionary rate of the immune traits, and to identify interesting atypical lineages (Weber

417 and Agrawal 2012). By investigating those exceptions, we may consequently focus on

418 documenting genetic mechanisms and adaptive value of different character states.

419 19

420

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533

534 APPENDIX

535 Literature review on peritoneal fibrosis in fish - Methods

536 To gather an overview of publication record encompassing peritoneal fibrosis in fish, we

537 performed two searches using Web of Science database on September 30, 2019. The first search

538 was oriented towards parasite-induced fibrosis in fish with terms: (parasit*) AND (fibro*) AND

539 ((teleost) OR (fish)). The other literature search was focused on fibrosis in fish in general while

540 we tried to avoid articles on human subjects: (fibrosis AND (fish OR teleost) NOT (human)).

541 We collected 1459 entries in total, of which 1337 articles were retained after double entries

542 removal. We considered an article to be suitable for our study if it was on ray-finned fish

543 (Actinopterygii) and contained information on any signs related to fibrosis, like organ adhesion,

544 spontaneous proliferation of fibroblasts (fibroma, fibrosarcoma), healing fibroplasia

545 (scarification), or encapsulation (of a parasite or an implant, for example), that could be inferred

546 from the title or article abstract. Encapsulation typically meant that an extra-bodily particle was

547 surrounded with a layer of fibroblasts and extracellular matter (e.g., collagen fibers). We then

548 sorted the suitable articles, by going into their main text, according to three criteria - the explicit

549 presence of fibrosis or capsule (“fibrosis only”, “capsule only”, “both”, “none”), its topology

550 (“viscera”, “other”, or “multiple”) and assumed cause (“parasite”, “toxicity”, “treatment”,

551 “tumor”, or “unknown”). For topology (location) classification, we took internal organs related to

552 excretory system, digestive tract, or reproduction (kidney, liver, gas bladder, gut, gonads,

553 including also peritoneum) as “viscera” and the remaining organs or tissues, such as gills, skin,

554 muscle, brain, heart, etc. as “other”. When tissues of both types were affected, we labelled the

555 article as “multiple”. We then grouped articles with respect to the given cause of the fibrosis-

556 related marks or encapsulation, where “parasite” was for infection with a uni- or multi-cellular

557 parasite, “toxicity” was when the study monitored known environmental pollution (e.g., heavy

558 metals), “treatment” was for deliberate manipulation with fish body or their living conditions

559 (e.g., adding estrogen into water to test the effect on male physiology), “tumor” was when

560 fibrosis happened spontaneously (e.g., fibrosarcoma) and “unknown” pooled studies where the

561 cause could not be identified. We extracted fish species names and sorted them into orders and

562 higher taxonomic categories according to the recent phylogenetic resolution of the Actinopterygii

563 tree of life by Hughes et al. (2018) and Rabosky et al. (2018). We then used this dataset to offer

564 an insight into the published literature on fish fibrosis.

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565 TABLES

566 Table 1. Species selected for the experimental test of peritoneal fibrosis response. We show the sample size, N, and average with doi: https://doi.org/10.1101/2020.07.08.191601 567 standard deviation (SD) for live weight before dissection in grams, Wtotal, in each experimental treatment. Note that Species #

568 corresponds to species number in Fig. 2. We also present data for two species that we did not use in the analysis as well as for the

569 stickleback that were kept in the treatment for 10 days (“10d”) indicated by “x”. The clown feather-back (Chitala ornata) were available undera

570 extremely fibrosed (level 3) before the experiment started. The kissing gourami (Helostoma temminckii) developed white spot

571 disease during the experiment. CC-BY-NC-ND 4.0Internationallicense ; Tapeworm this versionpostedFebruary4,2021. Species Higher taxonomic Control (PBS) Alum Common name Species Order homogenate Origin # rank N Wtotal N Wtotal N Wtotal 1 Senegal bichir Polypterus senegalus Polypteriformes Cladistia 6 3.98(0.75) 6 4.09(0.71) 6 4.09(0.72) captive-bred 2 common carp Cyprinus carpio Cypriniformes Otophysa 7 1.87(0.34) 7 1.72(0.46) 7 1.68(0.33) captive-bred 3 zebrafish Danio rerio Cypriniformes Otophysa 6 0.29(0.04) 7 0.29(0.08) 7 0.28(0.06) captive-bred 4 channel catfish Ictalurus punctatus Siluriformes Otophysa 7 2.24(0.53) 7 2.28(0.62) 7 2.15(0.39) captive-bred 5 Mexican tetra Astyanax mexicanus Characiformes Otophysa 6 0.82(0.17) 6 0.90(0.16) 6 0.91(0.08) captive-bred 6 bleeding-heart tetra Hyphesobrycon erythrostigma Characiformes Otophysa 5 0.97(0.24) 6 0.98(0.36) 6 1.09(0.21) wild .

7 rainbow trout Oncorhynchus mykiss Salmoniformes Protacanthopterygii 5 4.53(0.88) 5 4.24(1.03) 5 4.21(1.00) captive-bred The copyrightholderforthispreprint 8 pajama cardinalfish Sphaeramia nematoptera Kurtiformes Gobiaria 5 2.22(0.09) 5 2.21(0.45) 5 2.30(0.68) captive-bred 9 peacock gudgeon Tateurndina ocellicauda Gobiiformes Gobiaria 5 0.54(0.09) 4 0.68(0.11) 4 0.57(0.03) captive-bred 10 green chromis Chromis viridis Pomacentridae † Ovalentaria 4 1.37(0.11) 5 1.42(0.39) 5 1.35(0.35) wild 11 jewelled blenny Salarias fasciatus Blenniiformes Ovalentaria 4 1.57(0.54) 5 1.32(0.50) 3 1.68(0.97) wild 12 turquoise killifish Nothobranchius furzeri Cyprinodontiformes Ovalentaria 6 0.79(0.39) 6 0.77(0.38) 5 0.76(0.27) captive-bred 13 green swordtail Xiphophorus hellerii Cyprinodontiformes Ovalentaria 6 1.43(0.28) 6 1.29(0.16) 6 1.47(0.34) captive-bred

(which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade bioRxiv preprint

14 Nile tilapia Oreochromis niloticus Cichliformes Ovalentaria 7 2.18(0.69) 7 2.07(0.81) 7 1.95(0.70) captive-bred 15 Hosta stickleback Gasterosteus aculeatus Perciformes Eupercaria 6 0.31(0.24) 6 0.28(0.14) 6 0.30(0.16) wild x Hosta stickleback (10d) Eupercaria 6 0.36(0.14) 6 0.38(0.19) 6 0.38(0.17) wild doi: 15 a’ Bharpa stickleback Gasterosteus aculeatus Perciformes Eupercaria 6 0.27(0.05) 6 0.29(0.06) 6 0.30(0.13) wild https://doi.org/10.1101/2020.07.08.191601 x a’ Bharpa stickleback (10d) Eupercaria 5 0.37(0.07) 6 0.33(0.07) 5 0.30(0.03) wild 16 blue-gill sunfish Lepomis macrochirus Centrarchiformes Eupercaria 6 0.85(0.14) 6 0.79(0.18) 6 0.77(0.15) captive-bred 17 spotted green pufferfish Dichotomyctere nigroviridis Tetraodontiformes Eupercaria 3 1.49(0.27) 4 1.40(0.38) 4 1.29(0.36) captive-bred x clown feather-back Chitala ornata Osteoglossiformes Osteoglossomorpha 1 4.70 0 - 0 - captive-bred

x kissing gourami Helostoma temminckii Anabantiformes Anabantaria 5 4.56(0.78) 0 - 1 5.66 captive-bredavailable undera 572 † incertae sedis – the taxonomic position of the family within the higher taxonomic group is not well resolved CC-BY-NC-ND 4.0Internationallicense ; this versionpostedFebruary4,2021. . The copyrightholderforthispreprint

(which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade bioRxiv preprint

573 Table 2. Overview of the literature search for presence of peritoneal fibrosis in fish. We show fish phylogenetic group, the

574 presence of fibrosis and/or encapsulation, its location and cause. For brevity, we pooled less represented fish orders (with <6 doi:

575 articles) into “other” category and also grouped articles with species from more orders into “multiple”. Highlighted in gray are https://doi.org/10.1101/2020.07.08.191601

576 records of peritoneal fibrosis (column) and articles from Characiformes (row). Characiformes include the two tetra species that

577 did not respond with peritoneal fibrosis to any of the treatments in our immune challenge experiment. available undera

Fibrosis or capsule Position Cause Higher taxonomic peritoneal Order N rank fibrosis capsule fibrosis both none viscera other multiple parasite toxicity treatment tumor unknown only only Anabantaria Anabantiformes 9 6 1 2 4 4 1 4 5 CC-BY-NC-ND 4.0Internationallicense

Carangaria Pleuronectiformes 18 11 4 3 1 10 8 10 3 1 2 2 ; this versionpostedFebruary4,2021. Elopomorpha Anguilliformes 13 6 6 1 1 10 3 10 1 2 Eupercaria Centrarchiformes 12 4 5 3 10 1 1 12 Eupercaria Moronidae † 7 6 1 4 2 1 3 1 1 2 Eupercaria Perciformes 19 4 11 1 3 10 9 16 2 1 Eupercaria Spariformes 9 5 4 4 5 5 4 Eupercaria Sciaenidae † 8 5 3 2 5 1 5 2 1 Eupercaria Scorpaeniformes 6 3 2 1 3 3 3 1 1 1

Zeiogadaria Gadiformes 10 3 7 5 4 1 9 1 .

Otophysa Characiformes 12 2 8 2 7 5 8 1 1 2 The copyrightholderforthispreprint Otophysa Cypriniformes 56 23 19 5 9 32 24 26 5 17 8 Otophysa Siluriformes 26 21 3 2 21 5 11 1 12 2 Ovalentaria Cichliformes 11 7 1 1 2 8 3 2 3 4 2 Ovalentaria Cyprinodontiformes 12 8 2 1 1 6 6 5 1 6 Ovalentaria Mugiliformes 11 1 4 6 1 1 9 1 4 1 6 Pelagiaria Scombriformes 9 1 4 2 2 1 2 7 7 2 Protacanthopterygii Salmoniformes 50 29 10 3 8 1 31 19 20 2 13 7 8 30

(which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade bioRxiv preprint

other 53 26 14 3 10 1 37 13 3 26 6 11 9 1 multiple 24 9 6 1 8 1 13 9 2 11 4 1 7 1 TOTAL 375 175 116 23 61 7 220 144 11 197 29 81 53 15 doi: 578

† incertae sedis – the taxonomic position of the family within the higher taxonomic group is not well resolved https://doi.org/10.1101/2020.07.08.191601 available undera CC-BY-NC-ND 4.0Internationallicense ; this versionpostedFebruary4,2021. . The copyrightholderforthispreprint

579 FIGURE CAPTIONS

580 Figure 1. Peritoneal fibrosis in the experimental fish species. Individual points in the species plots

581 show recorded level of peritoneal fibrosis scored from their left flank (the side of the injection).

582 The fibrosis level was scored on an ordinal scale 0-3 and the jitter was used to show all data

583 points. Note that the three-spine stickleback (both populations of G. aculeatus) that stayed in the

584 experiment for 10 days are shown in grey as they did not enter data analysis. For completeness,

585 we also present data for two unused species in grey - kissing gourami (H. temminckii) and clown

586 feather-back (Ch. ornata). Full version of the abbreviated species names can be found in Table 1.

587 The G. aculeatus B is for population from Loch a’ Bharpa and G. aculeatus H for Loch Hosta.

588

589 Figure 2. Ancestral state reconstruction of species maximum peritoneal fibrosis. The values at the

590 branching nodes give estimates of the ancestral level of maximum peritoneal fibrosis level. Tree

591 structure and branch lengths are based on recent reconstruction of phylogeny of ray-finned fishes

592 (Actinopterygii) (Hughes et al. 2018). The squares of different intensity of purple colour show

593 average level of peritoneal fibrosis per treatment in each tested species. Treatment abbreviations

594 are Cont.: control (phosphate-buffered saline solution), TH: tapeworm antigen homogenate, A:

595 Alum vaccine adjuvant. Fish species drawings by M. F. Maciejewski (not to scale). Full version

596 of the abbreviated species names can be found in Table 1. The G. aculeatus B is for population

597 from Loch a’ Bharpa and G. aculeatus H for Loch Hosta.

598

599 Figure 1.

600 33

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601 Figure 2. doi: https://doi.org/10.1101/2020.07.08.191601 available undera CC-BY-NC-ND 4.0Internationallicense ; this versionpostedFebruary4,2021. . The copyrightholderforthispreprint

602